The present invention is generally related to liquid handling systems and in particular to systems for dispensing and aspirating of small volumes of reagents. It is particularly directed to a high throughput screening, polymerase chain reaction (PCR), combinatorial chemistry, microarraying, medical diagnostics and others. In the area of high throughput screening, PCR and combinatorial chemistry, the typical application for such a fluid handling system is in dispensing small volumes of the reagents, e.g. 1 ml and smaller and in particular volumes around 1 microliter and smaller. It is also directed to the aspiration of volumes from sample wells so that the reagents can be transported between the wells. The invention relates also to microarray technology, a recent advance in the field of high throughput screening. Microarray technology is being used for applications such as DNA arrays. In this technology the arrays are created on glass or polymer slides. The fluid handling system for this technology is directed to dispensing consistent droplets of reagents of submicroliter volume.
Development of instrumentation for dispensing of minute volumes of liquids has been an important area of technological progress for some time. Numerous devices for controlled dispensing of small volumes of liquids (in the range of 1 μl and smaller) for ink jet printing application have been developed over the past twenty five years. More recently, a wide range of new areas of applications has emerged for devices handling liquids in the low microliter range. These are discussed for example in “analytical chemistry” [A. J. Bard, Integrated chemical systems, Wiley-Interscience Pbl, 1994], and “biomedical applications [A. G. Graig, J. D. Hoheeisel, Automation, Series Methods in Microbiology, vol 28, Academic Press, 1999].
The present invention is also directed to medical diagnostics e.g. for printing reagents on a substrate covered with bodily fluids for subsequent analysis or alternatively for printing bodily fluids on substrates.
The requirements of a dispensing system vary significantly depending on the application. For example, the main requirement of a dispensing system for the ink jet applications is to deliver droplets of a fixed volume with a high repetition rate. The separation between individual nozzles should be as small as possible so that many nozzles can be accommodated on a single printing cartridge. On the other hand in this application the task is simplified by the fact that the mechanical properties of the liquid dispensed namely ink are well defined and consistent. Also in most cases the device used in the ink jet applications does not need to aspire the liquid through the nozzle for the cartridge refill.
For biomedical applications such as High Throughput Screening (HTS) the requirements imposed on a dispensing system are completely different. The system should be capable of handling a variety of reagents with different mechanical properties e.g. viscosity. Usually these systems should also be capable of aspiring the reagents through the nozzle from a well. On the other hand there is no such a demanding requirement for the high repetition rate of drops as in ink jet applications. Another requirement in the HTS applications is that cross contamination between different wells served by the same dispensing device be avoided as much as possible.
The most common method of liquid handling for the HTS applications is based on a positive displacement pump such as described in U.S. Pat. No. 5,744,099 (Chase et al). The pump consists of a syringe with a plunger driven by a motor, usually a stepper or servo-motor. The syringe is usually connected to the nozzle of the liquid handling system by means of a flexible polymer tubing The nozzle is typically attached to an arm of a robotic system which carries it between different wells for aspiring and dispensing the liquids. The syringe is filled with a liquid such as water. The water continuously extends through the flexible tubing into the nozzle down towards the tip. The liquid reagent which needs to be dispensed, fills up into the nozzle from the tip. In order to avoid mixing of the water and the reagent and therefore cross-contamination, an air bubble or bubble of another gas is usually left between them. In order to dispense the reagent from the nozzle, the plunger of the syringe is displaced. Suppose this displacement expels the volume ΔV of the water from the syringe. The front end of the water filling the nozzle is displaced along with it. The water is virtually incompressible. If the inner volume within the flexible tubing remains unchanged, then the volume ΔV displaced from the syringe equals the volume displaced by the moving front of the water in the nozzle. If the volume of the air bubble is small it is possible to ignore the variations of the bubble's volume as the plunger of the syringe moves. Thus the back end of the reagent is displaced by the same volume ΔV in the nozzle, and therefore the volume ejected from the tip is the same ΔV. This is the principle of operation of such a pump. The pump works accurately if the volume ΔV is much greater than the volume of the air bubble. In practice the volume of the air bubble changes as the plunger of the syringe moves. Indeed in order to eject a drop from the tip, the pressure in the tubing should exceed the atmospheric pressure by an amount determined by the surface tension acting on the drop before it detaches from the nozzle. Therefore at the moment of ejection the pressure in the tubing increases and after the ejection, it decreases. As common gasses are compressible, the volume of the air or gas bubble changes during the ejection of the droplet and this adds to the error of the accuracy of the system. The smaller the volume of the air bubble, the smaller is the expected error. In other words the accuracy is determined significantly by the ratio of the volumes of the air bubble and the liquid droplet. The smaller this ratio is the better the accuracy. For practical reasons it is difficult to reduce the volume of the air or gas bubble to below some one or two microliters and usually it is considerably greater than this. Therefore, this method with two liquids separated by an air or gas bubble and based on a positive displacement pump is not well suited for dispensing volume as low as 1 microliter or lower. There are also additional limitations on accuracy when submicroliter volumes need to be dispensed. For example, as the arm of the robotic system moves over the target wells, the flexible tubing filled with the water bends and consequently its inner volume changes. Therefore, as the arm moves, the front end of the water in the nozzle moves to some extent even if the plunger of the syringe does not. This adds to the error of the volume dispensed. Other limitations are discussed in Graig et al referred to above. Examples of such positive displacement pumps are shown in U.S. Pat. No. 5,744,099 (Chase et al). Similarly the problems of dispensing drops of small volume are also described and discussed in U.S. Pat. No. 4,574,850 (Davis) and U.S. Pat. No. 5,035,150 (Tomkins).
U.S. Pat. No. 5,741,554 (Tisone) describes another method of dispensing small volumes of fluids for biomedical application and in particular for depositing the agents on diagnostic test strips. This method combines a positive displacement pump and a conventional solenoid valve. The positive displacement pump is a syringe pump filled with a fluid to be dispensed. The pump is connected to a tubing. At the other end of the tubing there is a solenoid valve located close to the ejection nozzle. The tubing is also filled with the fluid to be dispensed. In this method the piston of the pump is driven by a motor with a well defined speed. This speed determines the flow rate of the fluid from the nozzle provided the solenoid valve is opened frequently enough and the duty cycle open/close of the valve is long enough. The solenoid valve is actuated with a defined repetition rate. The repetition rate of the valve and the flow rate of the pump determine the size of each drop. For example, if the pump operates at a flow rate of 1 μl per second and the repetition rate is 100 open-close cycles per second, then the size of each drop is 10 nl. However, for dispensing of submicroliter volumes for HTS applications this method is often inappropriate since it is required to aspire fluid through the nozzle in small quantities and then dispense it in fractions of this quantity. To avoid mixing of the fluid aspired with the one in the syringe pump, it is probably necessary to place a bubble of gas in the tube with the attendant problems described above. While this type of pump and solenoid valve is designed for dispensing series of drops of consistent size, it may not be well suited for dispensing single drops i.e. one drop on demand which is exactly the mode of dispensing used in the HTS applications. If the solenoid valve open time and/or operating frequency are too small for a given pump flow rate, the pressure in the dispenser will become too great, causing possible rupture or malfunctioning of the system.
U.S. Pat. No. 5,758,666 (Carl O. Larson, Jr. et al) describes a surgically implantable reciprocating pump having a floating piston made of a permanent magnetic material and incorporating a check valve. The piston can be moved by means of energising the coils in a suitable timing sequence. The piston allows the flow of liquid through it when it moves in one direction as the check valve is open and when it moves in the opposite direction, the check valve is closed and the liquid is pumped by the piston.
U.S. Pat. No. 4,541,787 (Sanford D. DeLong) describes an electromagnetic reciprocating pump with a “magnetically responsive” piston as it contains some ferromagnetic material. The piston is actuated by at least two coils located outside the cylinder containing the piston. The coils are energised by a current with a required timing.
Drops of microliter volume and smaller can be also generated by the method of electrospray which is mainly used for injection of a fluid into a chemical analysis system such as a mass spectrometer. In most cases the desired output of electrospray is not a stream of small drops but rather of ionised molecules. The method is based on supplying a liquid under pressure through a capillary towards its end and then a strong electrostatic field is generated at the end of the capillary by applying a high voltage, typically over 400V, between the end of the capillary and a conductor placed close to it. A charged volume of fluid at the end of the capillary is repelled from the rest of the capillary by Coulomb interaction as they are charged with the like charges. This forms a flow of charged particles and ions in the shape of a cone with the apex at the end of the capillary. A typical electrospray application is described in U.S. Pat. No. 5,115,131 (James W. Jorgenson et al).
There are inventions where the droplets emitted from a capillary are charged in order to prevent them from coming together with coagulation. This approach is described in U.S. Pat. No. 5,891,212 (Jie Tang et al) for fabrication of uniform charged spheres. U.S. Pat. No. 4,302,166 (Mack J. Fulwyler et al) teaches how to handle uniform particles each containing a core of one liquid and a solidified sheath. In this invention the electric field is applied in a similar way to keep the particles away from each other until the sheath of the particles has solidified. In this invention the particles are formed from a jet by applying a periodic disturbance to the jet. U.S. Pat. No. 4,956,128 (Martin Hommel et al) teaches how to dispense uniform droplets and convert these into microcapsules. A syringe pump supplies the fluid into a capillary. A series of high voltage pulses is applied to the capillary. The size of the droplets is determined by the supply of fluid through the capillary and the repetition rate of the high voltage pulses. The patent discusses generation of a single drop on demand. U.S. Pat. No. 5,639,467 (Randel E. Dorian et al) teaches a method of coating of substrates with a uniform layer of biological material. A droplet generator is employed which consists of a pressurised container connected to a capillary. A high constant voltage is applied between the capillary and the receiving gelling solution.
There are numerous methods for ink jet dispensing. The ink jet printing industry is the main driving force in the continuing progress in this field. Some of the well known methods are listed below:    a) One of the oldest methods of creating separated and uniform droplets is based on breaking a jet of liquid emerging from the nozzle. To control the breaking up of the jet into separated droplets periodical vibrations are applied to the jet of liquid. The optimal frequency F of such vibrations was estimated by Lord Rayleigh over a hundred years ago:
  F  =      V          4.51      ⁢                          ⁢      d      where                V—emerging jet velocity d—jet diameter.        
All droplets at this frequency are created uniformly with the same volume. A typical example of implementation of this method can be found in U.S. Pat. No. 5,741,554 (Tissone).    b) In numerous implementations of ink jet printing, pressure waves inside a liquid-holding chamber are created by a piezoelectric actuator. Accelerated by pressure waves, the liquid in the chamber achieves sufficient speed to move through the nozzle and to overcome capillary forces at the tip. In such a case a small droplet will be formed.    c) According to one method, the piezoelectric transducer changes the volume of the container and creates pressure waves in the liquid in the container. The action of compression wave causes some amount of the liquid (ink) to go through the nozzle and to form droplets which are separated from the bulk liquid in the container, see for example U.S. Pat. No. 5,508,726 (Sugahara).    d) In U.S. Pat. No. 5,491,500 (Inui) an ink jet head is described where liquid in the printing head is “pushed” by progressive waves created by a synchronized row of piezoelectric devices. Eventually, liquid in the printing head obtains enough speed to spray sequences of droplets through the nozzle.
In the methods b) to d) listed above it is necessary to have liquid without vapor and bubbles. Droplet viscosity, surface tension are very important. In the b) and c) cases droplets can be only of a fixed size.
In summary, the most common method of handling reagents used in HTS applications is based on a positive displacement pump and a gas bubble. The problem is that when dispensing volumes of reagents around 1 microliter or smaller the variation in the volume of the bubble during the dispensation compromises the accuracy. It has been found difficult to eject small droplets of precisely required volume using this method.
The use of a solenoid valve has two main disadvantages when used for HTS applications. The first one is the relatively high cost of a solenoid valve such that it cannot be a disposable element and thus cross contamination can be a major problem. Further difficulties have been experienced in achieving dead volumes smaller than 1 to 2 microliters in a conventional solenoid valve.
Piezo dispensers while used are often not well suited for dispensing reagents for medical applications. The reason is that the piezo dispenser commonly requires that fluid to be dispensed has well defined and consistent properties. Unfortunately, reagents and bodily fluids used in medical and biomedical applications have broadly varying properties and often contain particles and inhomogenities which can block the nozzle of the piezo dispenser.
As the size of wells becomes smaller and smaller, the problem of missing the correct well or dropping the liquid reagent at the wrong place of the substrate on which the reagent is being deposited becomes more and more significant. Measurement of the volume of the drops dispensed in the submicroliter range is a formidable task. It would be a highly desired and valuable feature of a liquid handling instrument to be capable of measurement of volume of individual droplets especially in the submicroliter range, and also measurement of the dispensation event which will allow excluding missing a drop.
U.S. Pat. No. 5,559,339 (Domanik) teaches a method for verifying a dispensing of a fluid from a dispense nozzle. The method is based on coupling of electromagnetic radiation which is usually light from a source to a receiver. As a droplet of fluid travels from the nozzle it obstructs the coupling and therefore the intensity of the signal detected by the receiver is reduced. The mechanism of such an obstruction is absorption of electromagnetic radiation by the droplet. The disadvantage of this method is that the smaller the size of the droplet, the smaller is the absorption in it. Almost certainly the method should not work for fluids which do not absorb the radiation.
For a range of applications such as high through put screening where minute droplets of fluids with a broad range of optical properties need to be dispensed the methods disclosed in this specification are inappropriate. Further the specification acknowledges that it will only operate satisfactorily with major droplets.